Cell-cell interactions are a feature of a variety of biological processes. In the activation of the immune response, for example, one of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury. The adhesion of leukocytes to vascular endothelium is an obligate step in their migration out of the vasculature (Harlan, Blood, 1985, 65, 515). As is well known in the art, cell-cell interactions are also critical for propagation of both B-lymphocytes and T-lymphocytes resulting in enhanced humoral and cellular immune responses, respectively.
In several instances, the adhesion of one cell type to another is mediated by interactions between specific proteins, termed "adhesion molecules," located on the plasma membrane of cells. The interaction between adhesion molecules is similar to classical receptor ligand interactions with the exception that the ligand is fixed to the surface of a cell instead of being soluble. One group of related (by peptide sequence), biologically significant molecules mediating cell-cell interactions are known in the art as CAMs (cellular adhesion molecules). CAMs include, for example, several intercellular adhesion molecules (i.e., ICAM-1, ICAM-2 and ICAM-3), endothelial leukocyte adhesion molecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1). The nucleotide and peptide sequences of PECAM-1 (platelet endothelial cellular adhesion molecule 1) indicate that it is also a member of the CAM family of genes. The CAM family is a part of the immunoglobulin superfamily of genes (Newman et al., Science, 1990, 247, 1219).
PECAM-1 is a member of the CAM family of proteins and is expressed on the surfaces of several cell types. Cells known to express PECAM-1 include, for example, endothelial cells, including human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC), glomerular endothelial (GEN) cells and cultured human pulmonary microvascular endothelial cells (HPMEC) ; platelets, monocytes, granulocytes, macrophages, some lymphocytes, some hematopoietic lineage cells and tumor cell lines from different mammalian species (see DeLisser et al., Immunol. Today, 1994, 15, 490.
A variety of evidence indicates that PECAM-1 is involved in cell-cell recognition events (DeLisser et al., Immunol. Today, 1994, 15, 490). For example, PECAM-1 mediates aggregation of L cells transfected with PECAM-1 cDNA (DeLisser et al., J. Biol. Chem., 1993, 268, 16307;) and, in certain T cell subsets, amplifies beta-1 integrin-mediated adhesion (Tanaka et al., J. Exp. Med., 1992, 176, 245). In vitro studies have implicated PECAM-1 in the initiation of endothelial cell contact (Albelda et al., J. Cell Biol., 1990, 110, 1227) and capillary tube formation (Merwin et al., unpublished results cited in Vaporciyan et al., Science, 1993, 262, 1580). In both in vitro and in vivo studies, PECAM-1 has been shown to be involved in the transmigration of white blood cells (e.g., neutrophils, monocytes, and other nucleated blood cells) through endothelial cell monolayers (Muller et al., J. Exp. Med., 1993, 178, 449). In addition to directly participating in cell-cell interactions, PECAM-1 apparently also regulates the activity and/or expression of other molecules involved in cellular interactions (Litwin et al., J. Cell Biol., 1997, 139, 219).
Due to PECAM-1's involvement in cellular processes preceding inflammation and tumorigenesis, it is believed that inhibitors of PECAM-1 expression may provide a novel therapeutic class of immunosuppressive and/or anti-inflammatory and anticancer agents with activity towards (1) autoimmune disorders such as multiple sclerosis, particularly autoimmune disorders of the thyroid such as Graves' disease, and undesired immune responses, such as, for example, those that occur in graft versus host disease (GVHD); (2) a variety of inflammatory diseases or disorders with an inflammatory component such as various forms of arthritis; allograft rejections; inflammatory diseases of the bowel, including Crohn's disease; various dermatological conditions such as psoriasis; corneal inflammation and the like, and (3) a variety of hyperproliferative diseases or disorders including, but not limited to, cancers, tumors, and the growth and spreading (metastasis) thereof.
To date, there are no known therapeutic agents which effectively prevent the expression of PECAM-1. Current agents which affect intercellular adhesion molecules include synthetic peptides, monoclonal antibodies, and soluble forms of the adhesion molecules. Synthetic peptides which block cellular interactions with PECAM-1 by mimicking a ligand of PECAM-1 have not been identified, although a peptidic mimic of PECAM-1 has been described (Liao et al., J. Exp. Med., 1997, 185, 1349). Monoclonal antibodies to PECAM-1 have been developed (Lastres et al., J. Immunol., 1994, 153, 4206) and may prove to be useful for the treatment of acute inflammatory responses or other conditions resulting from expression of PECAM-1 (Bogen et al., J. Exp. Med., 1994, 179, 1059; Murohara et al., J. Immunol., 1996, 156, 3550). However, with chronic treatment, the host animal can develop antibodies against the monoclonal antibodies themselves, thereby limiting their usefulness. In addition, monoclonal antibodies are large proteins which may have difficulty in gaining access to inflammatory sites. Soluble forms of the cell adhesion molecules suffer from many of the same limitations as monoclonal antibodies in addition to the expense of their production and their low binding affinity. All three of the above types of agents are polypeptides and are subject to proteolytic degradation in vivo, a feature which may limit the biological half-life and pharmacological effectiveness. Moreover, due to the high degree of homology between PECAM-1 and other members of the CAM family at the polypeptide level, agents which effect PECAM-1 activity might also effect other CAM proteins. Thus, there is a long felt need for molecules which effectively and specifically modulate PECAM-1 molecules.
Antisense oligonucleotides avoid many of the pitfalls of current agents used to block the effects of PECAM-1. For example, antisense oligonucleotides are smaller than monoclonal antibodies and most synthetic peptides and are thus expected to have better access to sites of inflammation. A variety of chemical modifications are known which serve to enhance the affinity of an antisense oligonucleotide for its target nucleic acid. Other chemical modifications have been described that render synthetic oligonucleotides more resistant to in vitro and in vivo degradation. Moreover, because of the degeneracy inherent in the genetic code, the homology between the nucleotide sequence of a nucleic acid which encodes a PECAM-1 protein and those nucleotide sequences encoding other CAM proteins will be less than the homology between the corresponding polypeptide sequences; as a result, antisense oligonucleotides have the potential to achieve a higher degree of specificity than many monoclonal antibodies. In short, the compounds of the invention are particularly effective for specifically modulating PECAM-1 molecules.